The development of efficient bioseparation processes for the production of high-purity biopharmaceutical products is one of the most pressing process development challenges facing the pharmaceutical and biotechnology industries today. Chromatography, by virtue of its high resolving power has made itself indispensable in downstream purification of biomolecules.
The last decade has seen a plethora of new stationary phase materials with a variety of base-matrix properties, linker arm chemistries and functional groups even for the same mode of chromatographic interaction. These chromatographic media differ in a variety of properties that are relevant to process applications such as capacities (dynamic, static and ionic), resolution, plate height, pressure drop, compressibility, protein recovery, operating flow-rate, chemical stability, and spacer arm and base-matrix chemistry.
During purification process development for biopharmaceuticals significant attention is paid to resin screening and selection by means of extensive empirical studies dealing mainly with selectivity, capacity and flow characteristics. Usually, not much attention is given to the possibility of transient pH changes at the resin screening stage. Nevertheless, such phenomena can have significant implications for the development of a scaleable and robust manufacturing process.
Perhaps, the most widely used chromatographic tool for preparative applications is ion exchange chromatography. pH of the mobile phase is a very important variable in ion exchange chromatography. In some cases, deliberate pH transitions have been exploited to develop more efficient purification processes. Retained pH gradients generated by either polyampholyte or simpler buffer systems have been utilized for chromatofocussing proteins into narrow bands. The ability of these pH gradients to separate protein mixtures has also been investigated.
Unintentional pH changes have also been reported in the literature. Under certain conditions, considerable pH excursions can occur in ion exchange systems even if the pH of the solution entering the column is the same as that with which the column was equilibrated. An increase in pH was observed on an SP Sephadex C-25 column when a column eluted with 1M ammonium acetate, pH 6.8 was re-equilibrated with the starting buffer (10 mM ammonium acetate, pH 6.8) (Karlsson et al., Ion-exchange chromatography. In Protein Purification: Principles, High-resolution methods and applications, Janson, J. C. and Ryden, L. (Eds.), Wiley-VCH, New York, 1998, pp 145-205). At the breakthrough of the 10 mM buffer, pH of the effluent was 7.5 and then it had gradually dropped to 6.8. A similar increase in pH (˜0.2 units) was reported by the same authors on a weak anion exchange column TSK-DEAE when a 10 mM phosphate buffer, pH 7.6 was replaced with 10 mM phosphate+1M sodium chloride at the same pH.
Helfferich and co-workers have made a detailed analysis of the pH behavior of buffer solutions in ion exchange columns using the wave theory of multicomponent equilibrium (Helfferich and Bennett, 1984, Reactive Polymers, 3:51-66). These phenomena have been explained on the basis of local ion exchange and dissociation phenomena between the charged ion exchanger and buffering species in solution. The three-component exchange of chloride, acetate and hydroxide ions has been used to predict some of these phenomena in anion exchange resins (Helfferich and Bennett, 1984, Solvent Extraction and Ion Exchange, 2(7&8):1151-184). Jansen et al. have modeled these phenomena by a complex model involving a thermodynamic model of equilibrium on ion exchange, Donnan potential and reaction equilibria to get more accurate predictions (Jansen et al., 1996, AIChE Journal, 42(7):1925-1937).
However, most of the observations in the literature have been made with dilute buffers at pHs at which they buffer weakly, hence the observations were not entirely unexpected. These studies did not investigate transient pH change phenomena using ion exchange media widely employed in the biotechnology industry or using buffers that buffer strongly at the pH of the experiments. Additionally, the contribution of the stationary phase backbone to these equilibria and their effect on transient pH phenomena was not been investigated. Accordingly, the direct impacts of these phenomena on process chromatography as practiced in the biopharmaceutical arena have not been clearly elucidated.
The present invention concerns the elucidation of the potential mechanisms behind such transient pH changes during chromatography processes, and provides practical methods to control their occurrence.